Spherical particles containing microorganism cells having enzyme activity

ABSTRACT

A process is presented for producing spherical particles containing microorganism cells having desired enzyme activity. The process comprises the steps of mixing the cells directly with a primary or secondary amine-containing polymer, combining the resulting mixture with an organic solvent to form a two-phase system, and then adding a bifunctional cross-linking agent to yield the spherical particles. The preferred enzyme activities are D-amino acid oxidase and glutarylacylase activities.

This application is a 371 of PCT/EP98/05729, filed Aug. 9, 1998, whichclaims priority to Austrian applications Ser. No. 1505/97, filed Sep. 9,1997, Ser. No. 1506/97, filed Sep. 9, 1997 and Ser. No. 1507/97, filedSep. 9, 1997.

The present invention relates to a process for inhibition of esteraseactivity in enzyme preparations, e.g. useful in enzymatic camlysedproduction of 7-aminocephalosporanic acid (7-ACA); and to the productionof enzyme preparations, e.g. useful in 7-ACA and/or7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid production.

7-ACA is a key intermediate in the production of e.g. pharmaceutically,active cephalosporin antibiotics, e.g. 7-ACA may be acylated at theamine group in position 7 of the ring system, e.g. a group known as avaluable group in the production of cephalosporin antibiotics or knownas a valuable group in an intermediate for their production; e.g. toobtain a 7-arninoacylated cephalosporin having a methylaceroxy group inposition 3 of the ring system, such as cefotaxime, cerpodoxime(proxetil), cephaloglycin, and in the production of other cephalosporinantibiotics or intermediates for their production which may be derivedfrom 7-ACA; e.g. by further reacting the methylacetoxy group in position3 of the ring system to obtain a 3-substituted cephalosporin substitutedby a group which is different from the acetoxymethyl group; e.g. a groupknown as a valuable group in the production of cephalosporin antibioticsor known as a valuable group in an intermediate for their production;e.g. by nucleophilic substitution of the aceroxy group, or e.g. bydeacerylation of the methylacetoxy group to obtain the hydroxymethylgroup (e.g. HACA); and e.g. further reacting a hydroxymethyl groupobtained in position 3 of the ring system, e.g. by nucleophilicsubstitution of the hydroxy group; or removal of the hydroxy function ofa hydroxymethyl group or removal of the hydroxymethyl group in position3 of the ring system: and, if desired, esterification of the carboxylicgroup in position 4 of the ring system, e.g. by a group known as avaluable group in the production of cephalosporin antibiotics or knownas a valuable group in an intermediate for their production: and, ifdesired, salt and/or solvent formation of a cephalosporin compoundobtained in such a reaction e.g. according to a conventional method.7-ACA and HACA may e.g. be obtained from cephalosporin C (Ceph C) bydeacylation of the amine group in position 7 of the ring system, anddeacetylation of the methylaceroxy group in position 3 of the ringsystem, respectively, e.g. enzymatically. An enzyme useful in enzymaticCeph C deacylation is e.g. D-amino acid oxidase (DAO) which catalysesthe oxydative desamination of Ceph C to formgiutaryl-7-aminocephalosporanic acid (G1-7-ACA) via the intermediatea-ketoadipoyl-7-aminocephalosporanic acid (KA-7-ACA). G1-7-ACA may behydrolised to obtain glutaric acid and 7-ACA, e.g. by a glutarylacylase(GAC). It is known that DAO and GAC containing enzyme preparations, suchas microorganism cells may contain esterase activity additionally.Undesired deacerylation of the side chain in position 3 of the ringstructure of the cephalosporin may occur, e.g. due to the presence ofesterase activity in enzymatically catalysed Ceph C deacylation, e.g.3-hydroxymethyl derivatives of Ceph C and G1-7-ACA may be formed. Thismay result in a significant decrease of 7-ACA quality and yield.Conventional methods for decreasing esterase activity present in thepresence of DAO activity by acetone or CuSO₄ treatment may decreaseesterase activity incompletely.

It was now surprisingly found that the treatment of a mixture havingesterase activity in the presence of DAO activity or having esteraseactivity in the presence of GAC activity with phenyimethyisulphonylfluoride (PMSF) may decrease esterase activity considerably, e.g.substantially complete, whereas DAO, or GAC activity, respectively mayremain high, e.g. substantially unchanged. This finding is surprisingbecause according to the present invention PMSF, known e.g. as anirreversible inhibitor of serine containing proteins by serinesulphonviation, may decrease esterase activity present in the presenceof DAO activity or in the presence of GAC activity selectively without,e.g. substantial influence on DAO or GAC activity; and even moresurprising is the selective and effective inhibition by PMSF of esteraseactivity present in the presence of GAC activity, e.g. because ofsimilar function and structure of acylases and esterases.

In one aspect the present invention provides a process for decreasing,e.g. substantially removing esterase activity present in the presence ofD-amino acid oxidase activity or in the presence of glutarylacylaseactivity in a mixture having esterase activity and D-amino acid oxidaseactivity and/or glutarylacylase activity, e.g. present in the form ofmicroorganism cells or in the form of a cell-free extract thereof, e.g.in the form of a cell-free extract thereof, comprising treating amixture having esterase activity and D-amino acid oxidase activityand/or glutarylacylase activity with phenyimethyisulphonyl fluoride,e.g. wherein D-amino acid oxidase activity or glutarylacylase activityremains, e.g. substantially the same after treatment withphenyimethyisulphonyl fluoride as before said treatment. TypicallyD-amino acid oxidase activity remains more than 91% of the originalvalue and in the case of glutarylacylase activity even more than 97% ofthe original value.

A process of the present invention may e.g. be carried out as follows:Known and e.g. commercially available microorganisms producing DAOactivity include e.g. Trigonopsis, Aspergillus, Penicillium, preferablyTrigonopsis variabilis microorganisms. Known and e.g. commerciallyavailable microorganisms producing GAC activity include e.g Pseudomonas,Achromobacter, Bacillus cereus or e.g. transformants, e.g. E. colitransformants, e.g. transformed according to e.g. a conventional method.A mixture having DAO activity or GAC activity and esterase activity maye.g. be obtained commercially or e.g. according to a conventionalmethod, and may be e.g. in the form of a cell-free extract, e.g. inimmobilised form, or in the form of microorganism cells, such as in theform of e.g. partly purified or impurified cells, and/or permeabilisedor non-permeabilised cells, and/or partly destroyed or intact cells,and/or immobilised or non-immobilised cells; such as obtainable from afermentation broth, e.g. according to a conventional method, preferablyin the form of a cell-free extract, e.g. immobilized. E.g. microorganismcells may be isolated, e.g. harvested from a fermentation broth and usedas such, e.g. in moist form, e.g. after centrifugation of thefermentation broth, or the cells may be further treated before or afterisolation from the fermentation broth, e.g. according to a conventionalmethod, such as homogenising cells to obtain, e.g. partly, destroyedcells, and/or permeabilising cells or fragments thereof to obtainpermeabilised cells or cell fragments and/or purifying cells or cellfragments to obtain e.g. partly purified cells and/or cell fragmentsand/or cell-free (e.g. by cell flocculation) extracts, and/orimmobilising cells or cell-free extracts to obtain immobilised cellsand/or immobilised cell fragments and/or immobilised cell-free extracts.E.g. immobilisation may be carried out according to a conventionalmethod; e.g. in the presence of acrylic (immobilisation) beads. such asEupergite® or in the presence of an ion exchange resin, e.g. such asRelite Dianion® according to a method described in the examples below;or e.g. according to another aspect of the present invention which isdescribed below.

The microorganism cells may be used in the form of an, e.g. buffered,aqueous cell suspension, obtainable e.g. by re-suspension of cells inwater or in a buffer solution after isolation from a fermentation broth,or in case of a cell-free extract an aqueous solution of the cell-freeextract may be used. The pH of a suspension or solution may be, e.g.approximately neutral, e.g. a pH of 6.5 to 8.5, such as 7.0 or around7.0 may be appropriate.

In a preferred embodiment of the present invention an esterase and DAOactivity having mixture may be e.g. substantially, free of e.g. nativecatalase activity, e.g. obtainable by treatment of an aqueous cellmixture having DAO, esterase and catalase activity in basic medium, e.g.in the presence of a caustic soda solution, e.g. at pH 9 to 11.5, suchas 10 to 11.

An esterase activity and DAO activity having mixture or an esteraseactivity and GAC activity having mixture, may be treated, e.g. incubatedat an appropriate temperature, e.g. at room temperature for anappropriate time, e.g. for several hours, such as for 1 to 5, e.g. 2.5to 4 hours or longer with PMSF, e.g. by addition of a PMSF solution,e.g. of an appropriate concentration range, e.g. of 0.5% to 25%, such as1% to 10% PMSF in an appropriate solvent, e.g. in alcohol, such as(C₁₋₄)alkanol, e.g. ethanol. Per 100 liters of a cell suspension anamount of e.g. 1000 ml of PMSF solution, e.g. in a concentration rangeas indicated above, may be sufficient to remove practically completelyand irreversibly undesired esterase activity, e.g. without, e.g.substantially decreasing the DAO or GAC activity present in the startingmixture. Removal of esterase activity may be determined by determinationof deacetylation products obtained in G1-7-ACA or 7-ACA reaction fromCeph C using a PMSF-treated DAO or GAC containing mixture. Forcomparison the reaction may be carried out using a non-PMSF treated DAOor GAC containing mixture under comparable reaction conditions.

A mixture may be obtained wherein DAO or GAC activity is high, e.g.substantially the same as before PMSF treatment and wherein esteraseactivity is, e.g. substantially, completely removed. A mixtureobtainable according to the present invention is useful, e.g. in theproduction of 7-ACA from e.g. Ceph C. Yields and purity of 7-ACAobtained may be improved if PMSF treated DAO and/or GAC activity is usedin comparison with the use of non-PMSF treated DAO and/or GAC activityunder comparable reaction conditions. Thus, e.g. the removal of esteraseactivity according to the present invention, results in less, e.g.substantially no, 3-deacetylation by-products which are due to esteraseactivity during DAO and or GAC reaction. The undesired amount of3-deacerylation by-products in G1-7-ACA or 7-ACA obtained by a processaccording to the present invention may be, e.g. substantially (typicallywithin 1% in a process giving 3% starting activity whereas when PMSF isnot used it may increase to 6%), the same as in Ceph C used as startingmaterial.

In another aspect the present invention provides a process for theproduction of glutaryl-7-aminocephaiosporanic acid comprising the steps

i) treating a mixture having esterase activity and D-amino acid oxidaseactivity with phenyimethyisulphonyl fluoride, e.g. to obtain a mixturewhich is substantially free of esterase activity, and e.g. havingsubstantially the same D-amino acid oxidase activity as before PMSFtreatment,

ii) reacting cephalosporin C with a mixture obtained in step i) toobtain glutaryl-7-aminocephalosporanic acid, and isolatinggiutaryl-7-aminocephalosporanic acid, if desired.

In another aspect the present invention provides a process for theproduction of 7-aminocephalosporanic acid comprising the steps

i) treating a mixture having esterase activity and D-amino acid oxidaseactivity with phenyimethyisulphonyl fluoride, e.g. to obtain a mixturewhich is substantially free of esterase activity, and e.g. havingsubstantially the same D-amino acid oxidase activity as before PMSFtreatment,

ii) reacting cephalosporin C with a mixture obtained in step i) toobtain glutaryl-7-amino-cephalosporanic acid,

iii) converting glutaryl-7-aminocephalosporanic acid into7-aminocephalosporanic acid, and isolating 7-aminocephalosporanic acidobtained, if desired.

Conversion of glutaryl-7-aminocephalosporanic acid into7-aminocephalosporanic acid may be carried out according to aconventional method, e.g. chemically or enzymatically, or e.g. accordingto another aspect of the present invention, e.g. as described herein.

In another aspect the present invention provides a process for theproduction of 7-amino cephalosporanic acid comprising the steps

i) treating a mixture having esterase activity and D-armino acid oxidaseactivity with phenyimethyisulphonyl fluoride, e.g. to obtain a mixturewhich is substantially free of esterase activity, and e.g. havingsubstantially the same D-amino acid oxidase activity as before PMSFtreatment,

ii) reacting cephalosporin C with a mixture obtained in step i) toobtain giutaryl-7-armino cephalosporanic acid,

iii) treating a mixture having esterase activity and glutarylacylaseactivity with phenyimethyisulphonyl fluoride, e.g. to obtain a mixturewhich is substantially free of esterase activity, and e.g. havingsubstantially the same glutarylacylase activity as before PMSFtreatment,

iv) reacting glutaryl-7-aminocephalosporanic acid obtained in step ii)with a mixture obtained in step iii) to obtain 7-aminocephalosporanicacid; and isolating 7-aminocephalosporanic acid obtained in step ii), ifdesired.

A process of the present invention to obtain 7-ACA from Ceph C may e.g.be carried our as follows: The starting material Ceph C may be e.g. inthe form of an aqueous suspension or solution of Ceph C in free-form orin salt form; e.g. Ceph C may be e.g. in the form of a Ceph C containingfermentation broth, e.g. wherein cells and solid have been removed, e.g.according to a conventional method, or in the form of a salt of Ceph C,e.g. in the form of a sodium salt. The suspension or solution of Ceph Cmay be treated with a mixture having DAO activity which is pre-treatedwith PMSF according to the present invention, e.g. in a form asdescribed above, e.g. an aqueous microorganism cell suspension or acell-free extract may be added to an aqueous suspension or solution ofCeph C (or vice versa); under introduction of oxygen into the suspensionor solution, e.g. in the form of oxygen gas or in the form of air, ormixtures thereof, e.g. if desired under pressure.

The reaction may be carried out at appropriate pH, e.g. at aroundneutral pH, e.g. at a pH of between 6.5 to 8.0, such as 7.0 to 7.5, atappropriate temperatures such as 10° C. to 30° C., e.g. at roomtemperature. The amount of DAO activity, e.g. the amount of a cellsuspension or of a cell-free extract having DAO activity in respect withthe amount of Ceph C is depending on the DAO activity present in thecell suspension used and on the amount of Ceph C to be reacted and isnot critical; an amount e.g. necessary for a short reaction time may beeasily determined e.g. by determination of the amount of G1-7-ACA formedin the mixture during, e.g. regular time intervalls by a conventionalmethod, e.g. by HPLC determination.

G1-7-ACA obtained may be isolated and purified, e.g. by a conventionalmethod or may be used as such for further processing, e.g. in theproduction of 7-ACA.

Oxidase activity may be determined as follows: 0.2 to 5 g of a mixturehaving DAO activity in the form of permeabilised cells is suspendedunder stirring in an aqueous 20 mM Ceph C solution at pH 8.0, at 25° C.under introduction of an of oxygen stream into the mixture to oxygensaturation.

The increase of G1-7-ACA and KA-7-ACA is determined by HPLC. Theactivity of “1 U oxidase” corresponds to the formation of 1 μmol/minG1-7-ACA and KA-7-ACA under the above conditions.

An aqueous solution or suspension of G1-7-ACA, e.g. obtainable orobtained according to the present invention may be brought into contactwith a mixture having GAC activity which is pretreated with PMSFaccording to the present invention, e.g. an aqueous, e.g. buffered cellsuspension or a cell-free extract may be added to an aqueous suspensionor solution of G1-7-ACA (or vice versa). The reaction may be carried outat appropriate pH, e.g. at slightly basic pH, e.g. including an pH ofbetween 7.5 to 8.5, such as around 8.2, at appropriate temperaturesincluding 10° C. to 30° C., e.g. room temperature. The amount of GACactivity in respect with the amount of G1-7-ACA is depending on the GACactivity present in the cell suspension or in the cell-free extract usedand on the amount of G1-7-ACA to be reacted and is not critical; anamount e.g. necessary for a short reaction time may be easily determinede.g. by determination of the amount of 7-ACA formed in the mixtureduring, e.g. regular time intervalls by a conventional method, e.g. byHPLC determination.

GAC activity may be determined as follows: 0.5 g to 2.0 g of a mixturehaving GAC activity are suspended under stirring in a 2% aqueousG1-7-ACA solution containing 5 mM phosphate and of a pH of 8.0 at 37° C.The pH is kept at 8.0 by addition of an aqueous 100 mM sodium hydroxidesolution. The amount of sodium hydroxide used is determined andcorresponds to the amount of glutric acid or 7-ACA, respectively formedfrom G1-7-ACA. The activity of “1 U acylases” corresponds to theformation of 1 μmol/min glutaric acid or 7-ACA, respectively under theabove conditions.

7-ACA obtained may be isolated and purified, e.g. by a conventionalmethod, or, if desired may be used as such, e.g. for further processing,e.g. in a 7-ACA reaction, e.g. according to a conventional method.

7-ACA obtained according to the present invention may be of high purity,e.g. 7-ACA obtained may contain a low amount of 3-deacetylation productsand may be obtained in high yields; e.g. in yields which are higher thanin a process wherein a non pretreated DAO activity and/or anon-pre-treated GAC activity is used under comparable reactionconditions.

It is known that e.g. enzyme containing microorganism cells may beimmobilised. Immobilised enzyme containing microorganism cells may beuseful e.g. because of the possibility of easy recovering and re-use.Known processes for the production of immobilized microorganism cellsmay have disadvantages, e.g. immobilised cells having poor enzymestability and/or low specific enzyme activity may be obtained, and/orthe immobilisation process may be complicated. Surprisingly a simpleimmobilisation process for microorganism cells having an enzymeactivity, such as DAO or GAC activity, was now found wherein immobilisedcells may be obtained in the form of solid spherical particles which maycontain stable and highly specific enzyme activity.

In another aspect the present invention provides a process for theproduction of spherical particles, e.g. in solid form, frommicroorganism cells having an enzyme activity, comprising the steps

i) treating microorganism cells with a primary or secondary aminecontaining polymer,

ii) contacting an organic solvent which is able to form a two-phasesystem with water with a mixture of microorganism cells, e.g. adding amixture of microorganism cells to a solvent which is able to form a twophase system with water,

iii) treating a mixture obtained in step ii) with a bifunctional agent;and isolating the spherical particles obtained;

e.g. further comprising

pre-treating the microorganism cells with a bifunctional reagent beforecarrying out steps i) or ii); and/or

adding a water-miscible solvent to a mixture obtained in step iii)before isolation of the spherical particles. and/or

adding a solid to a mixture obtained in step i) or to the microorganismcells before carrying out step i).

The process of the present invention is useful for immobilisingmicroorganism cells having an enzyme activity which are commonlydesignated as biocatalysts. Microorganisms which may be used accordingto the present invention include all types of microorganisms, such asbacterias, yeasts, funghi and actinomycetes, e.g. Trigonopsts, such asTrigonopsis variabilis, Agrobacterium, such as Agrobacteriumradiobacter, Rhodotoruia, such as Rhodotorula glutinis, Pleurotus, suchas Pleurotus ostreatus, Aspergillus, Penicillium, Pseudomonas,Achromobacter, Bacillus, such as Bacillus cereus, Schizosaccbaromyces,such as Schizosaccharomyces pombe; or e.g. transformants such as e.g.Eschericbia coli transformants having an enzyme activity. Enzymeactivity includes all types of enzyme activity, such as hydrolase,isomerase, lyase, decarboxylase, oxireductase activity. Examples ofmicroorganism cells having an enzyme activity includes e.g. Pleurotus,e.g. Pleurotus ostreatus cells having e.g. penicillin-V acylaseactivity, Agrobacterium, e.g. Agrobacterium radiobacter cells havinge.g. hydantoinase and/or N-decarbamoylase activity, Rhodotorula, e.g.Rhodotorula glutinis cells having e.g. esterase and/or oxidase activity,Trigonopsis, e.g. Trigonopsis variabilis having e.g. DAO activity,Schizosaccharomyces, such as Schizosaccharomyces pombe having e.g. GACactivity; and cells of transformants such as Eschericbia coli havinge.g. GAC and/or esterase activity.

The process of the present invention may be e.g. of particular interestin the production of immobilised microorganism cells which are useful in7-ACA or HACA production, e.g. starring from Ceph C, such as cellshaving DAO activity, e.g. in the presence of esterase (and/ordeacetyase) activity, and/or cells having GAC activity, e.g. in thepresence of esterase (and/or deacerylase) activity.

The process according to the present invention may be carried out asfollows: Microorganism cells having enzyme activity may e.g. be obtainedfrom a fermentation broth, e.g. according to a conventional method.Microorganism cells include cells in any form e.g. in the form of, e.g.partly purified or impurified cells, and/or permeabilised ornon-permeabilised cells, and/or, e.g. partly, destroyed or intact cells.E.g. microorganism cells may be isolated, e.g. harvested from afermentation broth and used as such, e.g. in moist form, e.g. aftercentrifugation of the fermentation broth, or the cells may be furthertreated before or after isolation from the fermentation broth, e.g.according to a conventional method, such as homogenising cells toobtain, e.g. partly, destroyed cells and/or permeabilising cells orfragments thereof to obtain permeabilised cells or fragments thereof,and/or purifying cells to obtain e.g. partly purified cells and/or cellfragments.

Undesired enzyme activity in microorganism cells may be removed beforeimmobilisation of the cells, e.g. according to a conventional method.E.g. catalase activity in Trigonopsis, e.g. Trigonopsis variabilis cellsmay e.g. be removed by treatment under a basic pH, e.g. a pH of 9 to 12,e.g. by addition of a caustic soda solution, e.g. in a mixture ofmicroorganism cells alone, or in a mixture of microorganism cells in thepresence of a primary and/or secondary amine containing polymer,optionally in the presence of additives, such as β-mercaptoethanol,ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA) for some time, e.g.ca. 15 minutes or longer.

According to the present invention the microorganism cells may bepre-treated with a bifunctional agent. A bifunctional agent as usedherein is to be understood as a compound having at least two reactivegroups which are able to react with primary and/or secondary aminegroups, including e.g. giutraldehyde, dimethyl pimelimidate,epichlorhydrin, N,N′-carbonyldiimidazole,1,4-butanediol-diglycidylether, maleic acid anhydride,dicyclohexylcarbodiimide, hexamethylenediisocyanate, preferablyglutraidehyde. Pre-treatment of the cells may be carried out in thefermentation broth, or after isolation of microorganism cells,preferably in the fermentation broth. E.g. the bifunctional agent may beadded to a microorganism cell suspension, e.g. in dissolved form, e.g.glutaraldehyde may be used in aqueous solution, e.g. in 10% to 30%aqueous solution and the mixture obtained after addition may be stirredfor some time, e.g. 30 minutes to some hours, such as 30 minutes to 5hours. The amount of the bifunctional agent in respect with the amountof cells is not critical; e.g. per 100 g of dry cell weight e.g. 0.1 gto 100 g, e.g. 5 g to 50 g of a bifunctional agent may be usedconveniently; e.g. if glutaraldehyde is used as a bifunctional agent 0.5g to 200 g, such as 1 g to 100 g of a 25% aqueous solution may beappropriate. After pre-treatment with a bifunctional agent the cells maybe harvested and isolated, e.g. according to a conventional method, suchas concentrated by microfiltration and/or centrifugation and stored in acool place, e.g. in frozen state. For further use frozen cells may bethawed and washed with spring water, and, if desired, concentrated, e.g.according to a conventional method such as microfiltration.

The microorganism cells, e.g. pre-treated as described above, may beused in the form of an, e.g. buffered, aqueous cell suspension e.g. byre-suspenion of cells in water after isolation from a fermentation brothand optional further treatment, e.g. at a pH of 6.0 to 10.0, such as 8.0or around 8.0. If desired, an additive, e.g. as conventional, such asβ-mercaptoethanol, ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA) maybe added to the suspension, e.g. in a conventional amount. To thesuspension obtained a primary and/or secondary amine containing polymerincluding e.g. a polyethylene amine and a polyvinyiamine, which ispreferably water-soluble, e.g. in aqueous solution, such as polyethyleneamine in e.g. 10% to 50% aqueous solution, e.g. having a molecularweight of 600,000 to 1,000,000 is added. Primary and/or secondary aminecontaining polymers are e.g. commercially available or e.g. may beproduced according to a conventional method. The amount of a primaryand/or secondary amine containing polymer in respect with the dry cellmass is not critical and includes e.g. an amount of 1 g to 100 g, suchas 5 g to 70 g, such as 10 g to 30 g per 100 g of dry microorganism cellweight

The cellipolymer mixture is contacted with an organic solvent which isable to form a two-phase system with water, e.g. the cell/polymermixture is introduced into an organic solvent which is able to form atwo-phase system with water. Appropriate organic solvents include e.g.trialkyl phosphates. such as tributyl phosphates, e.g. n-tributylphosphate. alkanes, e.g. n-hexane, n-heptane, aromatic hydrocarbons,e.g. toluene, natural or synthetic oils, e.g. soya oil, silicone oil,diesel oil, aromatic or aliphatic e.g. (C₃₋₈)alkyl, such as (C₄₋₆)alkyl,e.g. mono- or di- carboxylic acid alkyl, e.g. (C₁₋₈)alkyl, such as(C₁₋₄)alkyl, including e.g. glutaric, succinic, adipic acid dimethyl- ordiethylester, benzoic or phthalic acid ethyl-, butyl-, dibutyl- ordiisobutylester and anise alcohol, preferably trialkyl phosphate. Theamount of organic solvent may be such that the microorganism cellsupension may be dispersed in the organic solvent, e.g. by, e.g.laminar, stirring into the solvent; per g of dry cell weight e.g. 1 mlto 100 ml. such as 3 ml to 50 ml of solvent may conveniently be used.

The mixture containing the cells, the polymer and the organic solvent isstirred for some time, e.g. until a homogeneous supension is obtainedand treated with a bifunctional agent, including e.g. glutaraidehyde,epichlorohydrin, N,N′-carbonyidiimidazoie,1,4-butanediol-digiycidylether, maleic acid anhydride,dicyclohexylcarbodiimide, hexamethylenediisocyanate, preferablygiutaraidehyde. If a pre-treatment of the microorganism cells with abifunctional agent was carried out, the same bifunctional agent mayconveniently be used as in the pre-treatment. The amount of thebifunctional agent is not critical and includes an amount of e.g. 0.5 gto 100 g of a bifunctional agent per 100 g of dry cell weight, e.g. incase of using glutaraldehyde as a bifunctional agent e.g. 1 g to 200 gof a 5% aqueous solution may conveniently be used. Upon addition of thebifunctional agent to the mixture containing the cells. the polymer andthe organic solvent crosslinking may start and may be terminated e.g.within a few minutes and a suspension of, e.g. solid spherical particlesof the microorganism cells in the solvent mixture used may be obtained.If desired, the suspension obtained may be contacted with an organicsolvent which is water-miscible and which may be harmless in respectwith enzyme activity, e.g. which may be non-damaging in respect withenzyme activity, including e.g. alcohols, ketones, polyethylene glycols,glycerol, preferably glycerol, e.g. the cell suspension obtained may beintroduced into the organic solvent or the solvent may be introducedinto the cell suspension obtained. The amount of the water-miscibleorganic solvent is not critical and includes an amount which issufficient to form a liquid-liquid two phase system in the reactionmixture; e.g. per 100 g of dry cell weight conveniently e.g. 100 g to5000 g and more, e.g. 500 g to 5000 g of a water-miscible organicsolvent may be used. Phase separation, e.g. immediate, may occur uponcontact of the solvent with the cell suspension. Spherical cellparricles, e.g. solid. formed may be in the lower aqueous phaseobtained. The upper phase, e.g. containing mainly the organic solventwhich is able to form a two phase system with water may be separatedeasily and, e.g. substantially completely from the lower phase and maybe re-used, e.g. in a further immobilisation process. The spherical cellparticles obtained may be in solid form and may be isolated form theorganic-miscible-solvent phase, e.g. by a conventional method, e.g. bypouring the suspension through a vessel with perforated bottom,centrifugation, filtration. The isolated spherical particles may befreed from solvent residues, e.g. washed, e.g. with spring water.

Prior to crosslinking, i.e. prior to addition of the bifunctional agent,e.g. prior to addition of a solvent which is able to form a two-phasesystem with water, a solid, including e.g. aluminium oxide, activatedcarbon, bentonite may be added to the mixture of cells. The amount of asolid is not critical; conveniently an amount of 5 g to 200 g, such as50 g to 150 g per 100 g dry cell weight may be used. After suspension inthe solvent which is able to form a two phase system with water andcrosslinking by addition of the bifunctional agent into such a mixture,spherical cell particles may be obtained which may particularly easy beseparated and which may have a favourable narrow size-distribution-rangeand excellent mechanical stability.

The biocatalysts, e.g. spherical microorganism cell particles, e.g. insolid form, obtained according to the present invention may be storedfor later use e.g. in a cool place, e.g. in an appropriate buffer ore.g. in frozen form or lyophilised form.

Spherical particles containing microorganism cells which contain enzymeactivity obtainable, e.g. obtained, according to the present inventionare novel.

In another aspect the present invention provides spherical particlescontaining microorganism cells, e.g. in solid form, which contain enzymeactivity obtainable, e.g. obtained, from a process comprising the steps

i) treating microorganism cells having enzyme activity with a primary orsecondary amine containing polymer,

ii) contacting an organic solvent which is able to form a two-phasesystem with water with a mixture of microorganism cells, e.g. aftercarrying out step i), e.g. by addition of a mixture of microorganismcells to a solvent which is able to to form a two-phase system withwater,

iii) treating a mixture obtained in step ii) with a bifunctional agent;and isolating the spherical particles obtained;

e.g. further comprising

pre-treating the microorganism cells with a bifunctional reagent beforecarrying out steps i) or ii); and/or

adding a water-miscible solvent to a mixture obtained in step iii)before isolation of the spherical particles; and/or

adding a solid to a mixture obtained in step i) or to microorganismcells before carrying out step i).

Spherical particles, e.g. solid, having an enzyme activity obtainedaccording to a process of the present invention may be useful inenzymatic reactions, e.g. spherical particles, e.g. solid, having DAOactivity, e.g. (pre-)treated with PMSF and spherical particles, e.g.solid having GAC activity, e.g. (pre-)treated with PMSF may be useful inthe production of 7-ACA and/or G1-7-ACA and/or HACA.

In another aspect the present invention provides a process for theproduction of glutaryl-7-aminocephalosporanic acid comprising the steps

i) treating microorganism cells having D-amino acid oxidase activitywith a primary or secondary amine containing polymer,

ii) contacting an organic solvent which is able to form a two-phasesystem with water with a mixture of microorganism cells, e.g. aftercarrying out step i), e.g. by addition of a mixture of microorganismcells to a solvent which is able to to form a two-phase system withwater,

iii) treating a mixture obtained in step ii) with a bifunctional agent;and isolating the spherical particles obtained, and if desired, treatinga mixture having esterase activity in the presence of D-amino acidoxidase activity with phenyimethyisulphonyl fluoride, e.g. before stepiii), e.g. before step i); or treating spherical particles obtained instep iii) having esterase activity in the presence of D-amino acidoxidase activity with phenyimethyisulphonyl fluoride,

iv) reacting cephalosporin C with spherical particles obtained in stepiii) to obtain glutaryl-7-aminocephalosporanic acid, and isolatingglutaryl-7-aminocephalosporanic acid, if desired; e.g. and furtherconverting glutaryl-7-aminocephalosporanic acid obtained in step iv)into 7-aminocephalosporanic acid.

In another aspect the present invention provides a process for theproduction of giutaryl-7-aminocephalosporanic acid which comprisesreacting cephalosporin C with spherical particles obtainable by stepiii) to obtain glutaryl-7-aminocephalosporanic acid, and isolatingglutaryl-7-aminocephalosporanic acid, if desired.

Conversion of glutaryl-7-aminocephalosporanic acid obtained in step iv)into 7-aminocephalosporanic acid may e.g. be carried out according to aconventional method, e.g. chemically or enzymatically, or according toanother aspect of the present invention, e.g. using spherical particleshaving GAC activity obtainable or obtained as described above accordingto the present invention. If according to the present inventionspherical particles are used having DAO enzyme activity in the presenceof deacerylase activity; and/or having GAC activity in the presence ofdeacerylase activity, HACA may be obtained in a reaction with Ceph C orG1-7-ACA.

In another aspect the present invention provides the use of a processfor the production of spherical particles according to the presentinvention or the use of spherical particles produced according to thepresent invention in the production of7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid from cephalosporin Cor from glutaryl-7-aminocephalosporanic acid.

7-ACA or HACA produced according to the present invention may be usefulas an intermediate in the production of a cephalosporin, e.g. acephalosporin antibiotic or another intermediate in the productionthereof, e.g. by

acylating the amine group in position 7 of the ring system

further reacting the acetoxymethyl group in position 3 of the ringsystem

esterification of the carboxy group in position 4 of the ring system

salt and/or solvent formation,

e.g. as described above.

In another aspect the present invention provides a process for theproduction of 7-aminocephalosporanic acid comprising the steps

i) treating microorganism cells having giutarviacyiase activity with aprimary or secondary amine containing polymer,

ii) contacting an organic solvent which is able to form a two-phasesystem with water with a mixture of microorganism cells,

iii) treating a mixture obtained in step ii) with a bifunctional agent;and isolating the spherical particles obtained,

iv) if desired, treating a mixture having esterase activity in thepresence of glutarylacylase activity with phenyimethyisulphonylfluoride, e.g. before step iii), e.g. before step i) if desired; ortreating spherical particles obtained in step iii) having esteraseactivity in the presence of glutarylacylase acylase withphenyimethyisulphonyl fluoride, if desired,

v) reacting glutaryl-7-aminocephaiosporanic acid with sphericalparticles obtained in step iii) or obtained in step iv) to obtain7-aminocephalosporanic acid; and isolating 7-aminocephaiosporanic acidobtained, if desired.

In another aspect the present invention provides a process for theproduction of a cephalosporin, e.g. a cephalosporin antibiotic or anintermediate in its production, wherein 7-aminocephalosporanic acid or7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid, e.g.7-aminocephalosporanic acid, produced according to the present inventionis used as an intermediate.

“Substantially” as used herein means e.g. greater than 95%, e.g. 96%,97% of the original value or less than 5%, e.g. 4%, 3% of the originalvalue as appropriate.

In the following examples all temperatures are given in degrees Celsius.The following abbreviations are used:

7-ACA: 7-aminocephalosporanic acid

Ceph C: Cephalosporin C

DAO D-amino acid oxidase

GAC: glutarylacylase

GDA: glutaraldehyde

GA-7-ACA: Glutaryl-7-aminocephalosporanic acid

KA-7-ACA: α-ketoadipoyl-7-aminocephalosporanic acid

PEI Polyethylene imine

PMSF: Phenyimethyisulphonyl fluoride

TBP: n-Tributyl phosphate

The activity of “1 U oxidase” corresponds to the formation of 1 μmol/minG1-7-ACA and KA-7-ACA in an oxygen saturated 20 mM aqueous Ceph Csolution at pH 8 and 25° C. The activity of “1 U acylase” corresponds tothe formation of 1 μmol/min glutaric acid or 7-ACA in a 2% aqueousG1-7-ACA solution at pH 8 and 37° C.

EXAMPLE 1 General Procedure to Obtain a Mixture Having DAO ActivityWhich is Substantially free of Esterase Activity

From an aqueous fermentation broth having esterase activity and DAOactivity in water, a cell pellet is obtained from harvested cellsaccording to a conventional method (see e.g. Kubicek-Pranz, E. M. etal., Can. J. Microbiol. 1985, 31, pp 624-628), 10 g of the cell pelletobtained are re-suspended in 50 ml of water and treated for ca. 90minutes at a pH of ca. 11 with a caustic soda solution at roomtemperature to remove catalase activity. The pH of the mixture isadjusted to ca. 7 by addition of phosphoric acid.

To the mixture obtained 500 μl of an ethanolic solution containing 10 mgof PMSF/ml ethanol are added and the mixture obtained is incubated atroom temperature for ca. 3 hours. The cell suspension obtained may beused as such in a reaction which requires DAO activity.

DAO activity of cell pellet: 530 U oxidase;

DAO activity after caustic soda treatment: 490 U oxidase;

DAO activity after PMSF treatment: 450 U oxidase (=92% of activitybefore PMSF treatment).

EXAMPLE 2

A PMSF-treated cell suspension having DAO activity obtained as describedexample 1 is added to a solution of 10 g Ceph C in 1000 ml of water andthe pH of the solution obtained is adjusted to 7.5.

The mixture obtained is stirred for ca. 180 minutes at room temperatureunder introduction of oxygen into the mixture.

Yield of G1-7-ACA: 91%. Sum of 3-deacerylation products in the startingCeph C solution: 3% relative to Ceph C; Sum of 3-deacetylation productsin reaction solution obtained: 3% relative to starting Ceph C.

Example 2 is repeated but using a DAO activity without PMSF treatment

Yield of G1-7-ACA: 88%. Sum of 3-deacetylarion products in the starringCeph C solution: 3% relative to Ceph C; Sum of 3-deacetylation productsin reaction solution obtained: 6% relative to starting Ceph C.

EXAMPLE 3

A cell suspension obtained as described in example 1 is homogenisedbefore PMSF treatment (DAO activity in nomogenised cells: 385 U oxidase)and the homogenised cells obtained are treated with PMSF as described inexample 1 (DAO activity in homogenised and PMSF treated cells: 372 Uoxidase corresponding to 96% of activity before PMSF treatment). Thecell suspension obtained is reacted with Ceph C as described in example1.

Yield of G1-7-ACA: 93%; Sum of 3-deacerylation products in starting CephC solution: 3% relative to Ceph C; Sum of 3-deacerylation products inreaction solution obtained: 3% relative to starting Ceph C.

Example 3 is repeated but using a DAO activity without PMSF treatment.

Yield of G1-7-ACA: 89%. Sum of 3-deacetyiation products in starting CephC solution: 3% relative to Ceph C; Sum of 3-deacetylation products inreaction solution obtained: 7% relative to starting Ceph C.

EXAMPLE 4

10 g of a cell peliet obtained as described in example 1 before causticsoda treatment are suspended in 50 ml of water, 2 g of polyethyleneimine imolecular weight 600,000-1,000,000) are added to the supensionobtained and the mixture obtained is incubated for 90 minutes at pH 11(adjusted by caustic soda solution addition) at room temperature. The pHof a mixture obtained is adjusted to 8.5 by addition of phosphoric acid,and the suspension obtained is stirred with 50 ml of toluene. Anemulsion of fine droplet-like cell particles in solvent is obtained towhich 8 ml of 25% GDA solution are added. The fine dropiet-like cellparticles solidify to form solid spherical particles. The solidspherical particles are isolated and washed with spring water. DAOactivity in spherical particles: 290 U oxidase

The solid spherical particles obtained are suspended in 50 ml of 20 mMphosphate buffer pH 7.0, mixed with 500 μl of PMSF solution analogouslyas described in example 1 and the mixture obtained is incubated for 180minutes. DAO activity in PMSF treated solid spherical particles: 305 Uoxidase.

The solid PMSF treated spherical particles obtained are used in a Ceph Creaction analogously as described in example 2.

Yield of G1-7-ACA: 92%; Sum of 3-deacetylation products in starting CephC solution: 3% relative to Ceph C; Sum of 3-deacervlarion products inreaction solution obtained: 3% relative to starting Ceph C.

Example 4 is repeated but using solid spherical particles without PMSFtreatment.

Yield of G1-7-ACA: 89%. Sum of 3-deacerylation products in starting CephC solution: 3% relative to Ceph C; Sum of 3-deacerylation products inreaction solution obtained: 7% relative to starting Ceph C.

EXAMPLE 5

10 g of a moist cell pellet of the recombinant E. coli strain, e.g. CCM4229 (GAC activity: 720 U acylase) isolated from the fermentation brothare washed and re-suspended in 50 ml of 50 mM phosphate buffer pH 7.0.To the suspension obtained 0.5 ml of an ethanolic solution containing 10mg PMSF/ml, are added and the mixture obtained is stirred for ca. 3hours at room temperature. GAC activity: 708 U acylase (98% of activitybefore PMSF treatment).

10 g of G1-7-ACA are dissolved in 1000 ml of water and the pH isadjusted to 8.2. The PMSF-treated E. coli suspension obtained asdescribed above is added and the mixture obtained is stirred for ca. 180minutes at 12° whilst maintaining the pH at ca. 8.0-8.2.

Yield of 7-ACA: 93%; Sum of 3-deacetylation products in startingG1-7-ACA solution: 3% relative to G1-7-ACA; Sum of 3-deacervlarionproducts in reaction solution obtained: 3% relative to startingG1-7-ACA.

Example 5 is repeated but using GAC activity without PMSF treatment.

Yield of G1-7-ACA: 90%. Sum of 3-deacetylation products in startingG1-7-ACA solution: 3% relative to G1-7-ACA; Sum of 3-deacetylarionproducts in reaction solution obtained: 7% relative to startingG1-7-ACA.

EXAMPLE 6

10 g of a moist washed cell pellet of the recombinant E. coli strain,e.g. CCM 4229 (GAC activity: 720 U acylase) are suspended in 50 ml of 50mM phosphate buffer pH 7.0 and homogenised under a pressure of 700 bar.The homogenised cells (GAC activity: 752 U acylase) are mixed withethanolic PMSF solution as described in example 5. GAC activity in PMSFtreated cells: 768 U acylase.

The suspension obtained is added to a G1-7-ACA solution and the reactionis carried out as described in example 5.

Yield of 7-ACA: 89%; Sum of 3-deacerylation products in starringG1-7-ACA solution: 3% relative to G1-7-ACA; Sum of 3-deacetyiationproducts in reaction solution obtained: 3% relative to startingG1-7-ACA.

Example 6 is repeated but using GAC activity without PMSF treatment

Yield of G1-7-ACA: 83%. Sum of 3-deacervlarion products in startingG1-7-ACA solution: 3% relative to G1-7-ACA; Sum of 3-deacerylationproducts in reaction solution obtained: 6% relative to startingG1-7-ACA.

EXAMPLE 7

Homogenised cells from 10 g of a moist washed cell pellet of recombinantE. coli strain, e.g. CCM 4229 cells (GAC activity 7350 U acylase) aresuspended in 50 ml of 50 mM phosphate buffer pH 7.0 and mixed with aflocculation agent (1 ml of Sedifloc® CL 900-18/40). The pH of themixture obtained is adjusted to 5.2 by addition of acetic acid and themixture obtained is incubated for ca 1 hour at ca. 40° under stirringand stirred ca. for a further hour at 10°. The mixture obtained iscleared by centrifugation and the GAC-containing cell-free supernatant(GAC activity: 6150 U acylase) is adjusted to pH 7.5, mixed with PMSFsolution and incubated analogously as described in example 5. GACactivity of the cell-free extract obtained after PMSF treatment: 6220 Uacykase.

The mixture obtained is mixed with 10 g of acrylic (immobilisation)beads (Eupergit® C), treated with 50 ml of an aqueous 1 mol sodiumsulphate solution and incubated for 64 hours at room temperature undershaking. The enzyme-charged carrier material obtained is separated fromthe immobilisation solution and used as catalyst in a reaction with aG1-7-ACA solution analogously as described in example 5.

Yield of 7-ACA: 90%; Sum of 3-deacetylation products in startingG1-7-ACA solution: 3% relative to G1-7-ACA; Sum of 3-deacerylationproducts in reaction solution obtained: 3% relative to startingG1-7-ACA.

Example 7 is repeated but using immobilised GAC activity without PMSFtreatment.

Yield of G1-7-ACA: 83%. Sum of 3-deacerylation products in startingG1-7-ACA solution: 3% relative to G1-7-ACA; Sum of 3-deacetylationproducts in reaction solution obtained: 6% relative to startingG1-7-ACA.

EXAMPLE 8

GAC activity is immobilised analogously as described in example 7, butPMSF treatment is not carried out after immobilisation but duringimmobilisation by addition of 1 ml of PMSF solution analogously asdescribed in example 5 to a mixture containing the GAC-containingcell-free supernatant, Eupergit and sodium sulphate.

The enzyme-charged PMSF treated carrier material obtained is separatedfrom the immobilisation solution and used as a catalyst in a reactionwith a G1-7-ACA solution analogously as described in example 5.

Yield of 7-ACA and deacerylation products are substantially as describedin example 7.

EXAMPLE 9

GAC-containing supernatant is produced analogously as described inexample 7, but without PMSF treatment (GAC activity: 5830 U acylase).The solution obtained is mixed with 40 g of an ion exchange resin(Relite® Diaion-HPA25L), the mixture obtained is adjusted to pH 7.5 andincubated for ca. 300 minutes at room temperature under shaking. The ionexchange resin obtained is isolated and washed (GAC activity: 1865 Uacylase).

The exchange resin obtained is suspended in 200 ml of 50 mM phosphatebuffer, 2 ml of the PMSF solution as used in example 5 are added and themixture obtained is incubated for ca. 180 minutes under stirring. GACactivity in the PMSF treated ion exchange resin: 1810 U acylasecorresponding to 97% of the activity in the catalyst before PMSFtreatment

The enzyme-charged PMSF treated ion exchange resin obtained is used as acatalyst in a reaction with a G1-7-ACA solution analogously as describedin example 5. Sum of 3-deacerylation products in starting G1-7-ACAsolution: 3% relative to G1-7-ACA Sum of 3-deacerylation products inreaction solution obtained: 3% relative to starting G1-7-ACA

EXAMPLE 10

a. Pre-treatment

Cells of Trigonopsis variabilis, e.g. ATCC 58536 (535 g moist weight,DAO activity: 25,970 U oxidase) are pre-treated in 2.7 kg of afermentation broth by stirring 21 g of an aqueous 25% GDA solution intothe fermentation broth during ca. 1 hour at room temperature and at pH7.5. The cells are harvested from the cell suspension obtained bycentrifugation and stored in a frozen state. The frozen cell mass isthawed, washed with spring water by means of micro-filtration andconcentrated to a volume of ca. 735 ml. DAO activity in the concentratedcell mass: 26,350 U.

b. Polymer Treatment

735 ml of the cell mass obtained as described in step a. (100 g cell dryweight) are suspended with :mercaptoethanol (5 mM), EDTA (2 mM) and 40 gof an aqueous 50% PEI solution, molecular weight 600,000-1,000,000 inwater (ca. 800 ml total volume) and stirred slowly for ca. 90 minutes atpH 11 which is adjusted by addition of a caustic soda solution at roomtemperature. The pH of the mixture obtained is adjusted to pH 9 byaddition of phosphoric acid. DAO activity: 24,360 U (94% of pre-treatedDAO activity obtained as described in example 10 a.

c. Crosslinking

To 3000 ml of TBP in a stirring vessel the cell/polymer mixture obtainedin step b. is added at room temperature under stirring. A homogeneousdispersion of the cellipolymer droplets in TBP is obtained within ca. 30minutes to which 40 g of an aqueous 25% GDA solution are added.Crosslinking starts immediately and is terminated after some minutes andthe cells are obtained in the form of solid spherical particles. 2000 gof glycerol are added to the mixture obtained at 15° to 20° and phaseseparation between the TBP phase and the aqueous phase is achieved. Thelower phase containing mainly glycerol and solid spherical cellparticles is separated from the upper phase containing mainly TBP. Theupper phase obtain ed (2900 th) may be re-used e.g. in anothercrosslinking bat ch. The solid spherical particles arc isolated from thelower phase after ca. 90 minutes and washed with spring water in orderto remove TBP and glycerol residues. 580 g of moist solid sphericalparticles having oxidase activity are obtained, corresponding to 121 gof dry weight Specific DAO activity in the solid spherical particles is32 U/g moist weight (153 U/g dry weight), i.e. total DAO activity is18,560 U (71% of the pre-treated DAO activity obtained as described inexample 10 a.

EXAMPLE 11

580 g of the solid spherical particles (moist weight) obtained inexample 10 c. are suspended in 2000 ml of spring water and stirred atroom temperature. 20 mg of PMSF are dissolved in 20 ml of absoluteethanol and the solution obtained i s added to the oxidase suspensionwithin ca. 2 minutes. The mixture is incubated for ca. 3 hours, thesolid spherical particles obtained are isolated and washed with aqueousethanol (1000 ml, 10%) and with spring water. 563 g of solid sphericalparticles (moist weight; corresponding to 138 g of dry weight) areobtained. Specific DAO activity in the PMSF treated solid sphericalparticles is 31 U/g moist weight (148 U/g dry weight), i.e. total DAOactivity is 17,450 U (67% of the pre-treated DAO activity obtained asdescribed in example 10 a. and 94% of the activity before PMSFtreatment). The esterase activity in the spherical particles obtained issubstantially removed.

EXAMPLE 12

The pH of a cell/polymer mixture produced as described in examples 10 a.and b. is adjusted to 9.0 by addition of phosphoric acid and 80 g ofaluminium oxide are added. Crosslinking is carried out with the mixtureobtained as described in example 10 c.

630 g of solid spherical particles (moist weight, corresponding to 193 gof dry weight) are obtained. Specific DAQ activity in the solidspherical particles is 26 U/g moist weight (85 U/g dry weight), i.e.total DAO activity is 16,440 U (63% of the pre-treated DAO activityobtained as described in ex ample 10 a.). A narrowsize-distribution-range of the particles of 240-500 μm is obtained.

EXAMPLE 13

Is carried out analogously as described in example 10 but withoutaddition of glycerol. After crosslinking the suspension is stirred forca. 60 minutes. The solid spherical particles obtained in TBP suspensionare isolated by running the suspension obtained through a vessel havinga perforated bottom, wherein the perforation holes are smaller than thesize of the solid spherical particles obtained. 2680 ml of TBP areobtained. TBP residues are removed from the solid spherical particlesobtained by washing with spring water.

610 g of solid spherical particles (moist weight, corresponding to 126 gof dry weight) are obtained. Specific DAO activity in the solidspherical particles is 36 U/g moist weight (174 U/g dry weight), i.e.total activity is 21,960 U (85% of the pre-treated DAO activity obtainedas described in example 10 a.

EXAMPLE 14

PMSF treated solid spherical particles obtained analogously as describedin examples 10 and 11 (81 g moist weight, DAO activity: 2,500 U) aremixed with 1 litre of a 75 mM aqueous Ceph C solution of pH 7.2. Airunder 5 bar pressure is introduced into the mixture obtained for ca. 80minutes. The solution is separated off from the solid sphericalparticles and is analysed by HPLC. 0.6 mM Ceph C, 70.7 mM G1-ACA and 3mM of KA-7-ACA are determined in the separated solution.

Example 14 is repeated 142 times each time using a fresh Ceph C-solutionand each time using the same PMSF treated solid spherical particlesdescribed above in example 14.

The solution is separated off from the solid particles in batch 142after ca. 160 minutes of contact with Ceph C and is analysed by HPLC.0.5 mM Ceph C, 69.8 mM G1-ACA and 0.4 mM of KA-7-ACA are determined inthe separated solution.

After batch 142 the solid spherical particles had been used for 308hours. 93 g of solid spherical particles (moist weight, corresponding to24 g of dry weight) are isolated.

Specific DAO activity in the solid spherical particles obtained is 12U/g moist weight corresponding to 47 U/g dry weight, i.e. total activityis 1,120 U (45% of the original activity).

EXAMPLE 15

Cells of Schizosaccharomyces pombe, e.g. ATCC 38399 and e.g. ATCC 38436(82 g and 90 g moist weight, respectively) having DAO activity are eachpre-treated (separately) analogously as described in example 10 a. with3.3 g of an aqueous 25% GDA solution and harvested and stored in afrozen state. After thawing the mixture is washed with spring water andconcentrated to obtain 20.4 g of dry substance in 164 ml from e.g. ATCC38399 and 24.1 g of dry substance in 180 ml from e.g. ATCC 38436.

7 g of cell dry weight of each of e.g. ATCC 38399 and e.g. ATCC 38436are treated (separately) with 2.8 g of an aqueous 50% PEI solution asdescribed in example 10 b.

Crosslinking is carried out with both suspensions obtained (separately)analogously as described in example 10 c. in 230 ml TBP under additionof 3.5 g of an aqueous 25% GDA solution and under addition of 400 g ofglycerol. The solid spherical particles obtained are washed with springwater over a sieve in order to remove glycerol and TBP residues.

59.5 g moist weight of solid spherical particles having DAO activity(14.0 g dry weight) are obtained from e.g. ATCC 38436 and 55.6 g moistweight of solid spherical particles having DAO activity (16.1 g dryweight) are obtained from e.g. ATCC 38399. The solid spherical particlesobtained show similar DAO reaction characteristics and similar stabilitycharacteristics as the spherical particles obtained as described inexample 10 c.

Schizosaccharomyces pombe is suitable for the cloning and expression ofvarious enzymes. Other biocatalysts, e.g. solid spherical particleshaving other enzyme activity may be obtained in a simple manneranalogously as described in the present example.

EXAMPLE 16

Cells of Pleurotus oszretzus (90 g moist weight, 18,970 U penicillin Vacylase) are pretreated with 3.3 g of an aqueous 25% GDA solutionanalogously as described in example 10 a. The cells from the cellsuspension obtained are harvested, stored in a frozen state, and afterthawing are washed with spring water and concentrated bymicrofiltration.

18.6 g of cells obtained (dry weight) are treated with 7.4 g of anaqueous 50% PEI solution. Cell crosslinking is carried out analogouslyas described in example 10 c. in 560 ml TEP under addition of 7.4 g ofan aqueous 25% GDA solution and under addition of 370 g of glycerol. Thesolid spherical particles obtained are washed with spring water througha sieve in order to remove glycerol and TBP residues.

223 g of solid spherical particles containing penicillin V acylase(moist weight corresponding to 29.4 g dry weight) are obtained. Specificpenicillin V acylase is 50.4 U/g moist weight, i.e. total penicillin Vacylase is 11,240 U (59% the original activity).

1 U penicillin V acylase corresponds to the formation of 1 μmol perminute of 6-aminopenicillanic acid in an aqueous phosphate-bufferedK-penicillin-V solution (50 g/l) at pH 7.5 and 28°.

EXAMPLE 17

Cells of Trigonopsis vartabilis, e.g. ATCC 58536 (54 g moist weight, DAOactivity: 2,600 U oxidase) are produced analogously as described inexamples 10 a. and 10 b. Crosslinking is effected analogously asdescribed in example 10 c., but a cell/polymer mixture containingTrigonopsis variabilis cells (ca. 10 g dry weight) is added understirring into either 300 ml of glutaric dimethylester or 300 ml ofadipic dimethylester (instead of 3000 ml of TBP to 100 g cell dryweight). Crosslinking in each of the mixtures obtained (separately) iscarried out under addition of 5 g of an aqueous 25% GDA solution to eachof the mixtures and under addition of 400 g of glycerol to each of themixture at a temperature of 15° to 20° analogously as described inexample 10 c.

93.4 g and 86.9 g, respectively of solid spherical particles having DAOactivity are obtained (moist weight, corresponding to 20.6 g and 20.2 gof dry weight, respectively).

Specific DAO activity is 14.8 U/g moist weight or 12.5 U/g moist weight,respectively, i.e. total activity is 1382 U (53% of the originalactivity) or 1086 U (42% of the original activity), respectively.

What is claimed is:
 1. A process for the production of sphericalparticles having an enzyme activity from microorganism cells having theenzyme activity, comprising the steps i) directly adding to themicroorganism cells a primary or secondary amine containing polymer, ii)mixing the treated microorganism cells from step i) with an organicsolvent to form a two-phase system with water, and iii) adding to themixture obtained from step ii) a bifunctional agent to yield thespherical particales with the enzyme activity.
 2. A spherical particlehaving an enzyme activity obtained from a process comprising the stepsi) directly adding to microorganism cells having the enzyme activity aprimary or secondary amine containing polymer, ii) mixing the treatedmicroorganism cells from step i) with an organic solvent to form atwo-phase system with water, and iii) adding to the mixture obtainedform step ii) a bifunctional agent to yield the spherical particles withthe enzyme activity.
 3. A spherical particle of claim 2 wherein theenzyme activity is D-amino oxidase activity or glutarylacylase activity.4. A spherical particle of claim 3 where the process further comprisesadding phenylmethylsulphonyl fluoride to the mixture obtained from stepii).